Control of a tyrosyl radical mediated protein cross-linking reaction by electrostatic interaction

Herein, we demonstrate the control of protein heteroconjugation via a tyrosyl coupling reaction by using electrostatic interaction. Aspartic acid and arginine were introduced to a tyrosine containing peptide tag (Y-tag) to provide electrostatic charge. Designed negatively or positively charged Y-tags were tethered to the C-terminus of Escherichia coli alkaline phosphatase (BAP) and streptavidin (SA), and these model proteins were subjected to horseradish peroxidase (HRP) treatment. The negatively charged Y-tags showed low reactivity due to repulsive interactions between the Y-tags with the negatively charged BAP and SA. In contrast, the positively charged Y-tags showed high reactivity, indicating that the electrostatic interaction between Y-tags and proteins significantly affects the tyrosyl radical mediated protein cross-linking. From the heteroconjugation reaction of BAP and SA, the SA with the positively charged Y-tags exhibited favorable cross-linking toward negatively charged BAP, and the BAP-SA conjugates prepared from BAP with GY-tag (GGGGY) and SA with RYR-tag (RRYRR) had the best performance on a biotin-coated microplate. Encompassing the reactive tyrosine residue with arginine residues reduced the reactivity against HRP, enabling the modulation of cross-linking reaction rates with BAP-GY. Thus, by introducing a proper electrostatic interaction to Y-tags, it is possible to kinetically control the heteroconjugation behavior of proteins, thereby maximizing the functions of protein heteroconjugates.     

T-type Ca2+ channel blockers prevent cardiac cell hypertrophy through an inhibition of calcineurin-NFAT3 activation as well as L-type Ca2+ channel blockers

T-type Ca2+ channels (TCCs) are involved in cardiac cell growth and proliferation in cultured cardiomyocytes. Underlying molecular mechanisms are not well understood. In this study, we investigated the role of TCCs in signal transduction in cardiac hypertrophy compared with L-type Ca2+ channels (LCCs). Cardiomyocytes dissociated from neonatal mouse ventricles were cultured until stabilization. Cell hypertrophy was induced by reapplication of 1% fatal bovine serum (FBS) following a period (24 h) of FBS depletion. Cell surface area increased from 862+/-73 microm2 to 2153+/-131 microm2 by FBS stimulation in control (250+/-1.8%). T-type Ca2+ current (I(CaT)) was inhibited dose-dependently by kurtoxin (KT) and efonidipine (ED) with IC50 0.07 microM and 3.2 microM, respectively in whole-cell voltage clamp. On the other hand, 1 microM KT which inhibits I(CaT) over 90% did not effect on L-type Ca2+ current (I(CaL)). 10 microM ED had the ability of I(CaL) blockade as well as that of I(CaT) blockade. 3 microM nisoldipine (ND) suppressed I(CaL) by over 80%. The increase in cell surface area following reapplication of FBS as observed in control (250+/-1.8%) was significantly reduced in the presence of 1 microM KT (216+/-1.2%) and virtually abolished in the presence of 10 microM ED (97+/-0.8%) and 3 microM ND (80+/-1.1%). Hypertrophy was associated with an increase in BNP mRNA of 316+/-3.6% in control and this increase was reduced as well in the presence of 1 microM KT (254+/-1.8%) and almost abolished in the presence of 10 microM ED (116+/-1.1%) and 3 muM ND (93+/-0.8%). Immunolabeling showed that translocation of nuclear factor of activated T cells (NFAT3) into the nucleus in response to FBS stimulation was markedly inhibited by either KT or ED as well as ND. Calcineurin phosphatase activity was upregulated 2.2-fold by FBS, but KT, ED and ND decreased this upregulation (1.7-fold, 0.8-fold, and 0.7-fold with KT, ED and ND respectively). These results suggest that blockade of Ca2+ entry into cardiomyocytes via TCCs may block pathophysiological signaling pathways leading to hypertrophy as well as via LCCs. The mechanism may be the inhibition of calcineurin-mediated NFAT3 activation resulting in prevention of its translocation into the nucleus.     

Ectodermal factor restricts mesoderm differentiation by inhibiting p53

During gastrulation of the amphibian embryo, specification of the three germ layers, endo-, ecto-, and mesoderm, is regulated by maternal and zygotic mechanisms. Although it is known that mesoderm specification requires the cooperation between TGF-beta signaling and p53 activity and requires maternal factors, essential zygotic factors have been elusive. Here, we report that the Zn-finger protein XFDL156 is an ectodermal, zygotic factor that suppresses mesodermal differentiation. XFDL156 overexpression suppresses mesodermal markers, and its depletion induces aberrant mesodermal differentiation in the presumptive ectoderm. Furthermore, we find that XFDL156 and its mammalian homologs interact with the C-terminal regulatory region of p53, thereby inhibiting p53 target gene induction and mesodermal differentiation. Thus, XFDL156 actively restricts mesodermal differentiation in the presumptive ectoderm by controlling the spatiotemporal responsiveness to p53.     

A pilot study of the relationship between bowel habits and sleep health by actigraphy measurement and fecal flora analysis

We have previously reported that there may be a relationship between bowel habits including functional constipation (FC) and irritable bowel syndrome and sleep health. However, our previous studies were based on only subjective parameters by self-reported questionnaire. The aim of this study is to investigate the relationship between bowel habits such as FC and sleep health using objective parameters. Sleep health was assessed by actigraphy measurement and bowel habits by fecal flora analysis. The FC and control subjects, whose bowel habits were defined at Rome II, were recruited from evaluated respondents in our previous study directed at middle-aged Japanese women, ten FC and ten control subjects participating in this study. Wake after sleep onset (WASO) and WASO (%) (WASO/total sleep time multiplied by 100) in FC subjects was significantly longer and greater than those in control subjects, respectively. Average activity during sleep in FC subjects was significantly higher than that in control subjects. FC had no effect on total sleep time. Bifidobacterium is broadly accepted to be useful intestinal bacteria for human health and one of the indices showing that the intestinal environment is in a desirable condition. Bifidobacterium counts per gram of wet feces and proportion in total bacterial cell counts in FC subjects were significantly lower than those in control subjects. In conclusion, these results suggest that corresponding to low Bifidobacterium counts and proportion, sleep in FC subjects may be worse than that in control subjects. There may be a relationship between bowel habits and sleep health. Bowel habits such as FC might be a risk factor for sleep disorders.     

Intracellular changes of metal elements by fucoidan extracted from brown seaweed (Cladosiphon okamuranus)

This study was undertaken to elucidate the intracellular changes of metal elements after the administration of fucoidan extracted from Cladosiphon okamuranus. TRL1215 cells (normal rat liver cell line) were treated with 0, 0.1, or 1.0 mg/ml fucoidan and incubated in 5% CO2 at 37 degrees C. The cellular levels of Mg, Al, Fe, and Zn were significantly increased in the 1.0 mg/ml fucoidan-treated cells compared to those of the 0.1 mg/ml fucoidan-treated cells and the control. Next, TRL1215 cells were cultured on Mylar film overnight. At 24 h after 5-bromo-2'-deoxyuridine dosing, 0, 0.1, or 1.0 mg/ml fucoidan was treated for 9 h. The cellular distribution of elements was analyzed using in-air micro-micro-particle induced X-ray emission. The X-ray spectra showed that yields of Al, Mg, and Zn were high in order of the 1.0 mg/ml fucoidan-treated sample, the 0.1 mg/ml fucoidan-treated sample, and the control. Fe yield was mildly increased by fucoidan administration. In fucoidan-treated cells, the focal accumulation of Br was correlated spatially with phosphorous-rich region, suggesting that Br was localized within the nucleus. Al distribution provided a spatial association with Br map. These data suggest that fucoidan increases the accumulations of Al, Mg, Fe, and Zn in normal rat hepatocytes, and fucoidan-binding Al is postulated to be transferred into the nucleus.     

Taenia taeniaeformis: fate and proliferation of mucosal cells during gastric hyperplasia in larvae infected rats

Fate and proliferation of gastric mucosal cells during hyperplasia of Taenia taeniaeformis eggs inoculated Wistar rats were investigated using PCNA immunohistochemistry, BrdU labeling and other histopathologic staining techniques. Results revealed marked cell proliferation in gastric corpus and antral mucosa of infected rats as evidenced by increased lengths of proliferative zones and indices of BrdU labeling. The gastropathy in corpus was characterized by massive accumulation of precursors, neck and intermediate cells following significant decreases in numbers of parietal and zymogenic cells. Gastropathy in antrum was described with significant increases in precursors and mucous cells. Our results suggested that T. taeniaeformis-induced gastric hyperplasia was initiated by depletion of parietal cells presumably due to the cestode's ES products. As a result, there was inhibition of zymogenic cell differentiation due to the disruption of normal development pathways of gastric mucosal lineages. These sequences of events were considered to cause the increase in cell proliferation and accumulation of intermediate cells resulting to the hyperplastic lesions.     

Taenia taeniaeformis: colonic hyperplasia in heavily infected rats

Only one study previously mentioned the involvement of colon during Taenia taeniaeformis larvae infection in rats with inconsistent occurrence of lesions. Present study aimed to determine the consistency of histopathologic changes in colonic epithelia, and the proliferation of mucosal cells through BrdU and PCNA immunohistochemistry. Results demonstrated that crypt hyperplasia of the colon was found in all infected rats, although variable in degree even in a single tissue section. Cystic cavities were frequently seen in severely hyperplastic mucosa. Proliferative zone lengths were significantly increased and PCNA positive cells were observed throughout the colonic crypt lengths at 9 but not at 6 weeks post infection. Cell proliferation involving the major types of cells in the epithelial colon was also increased in infected rats at 9 weeks post infection, with labeling indices significantly greater than the control rats throughout the BrdU time course labeling. Findings suggested that massive increases in epithelial cells and depth of colonic crypts were due to a remarkable increase in cell proliferation. The study concluded that enteropathy in the colon during T. taeniaeformis infection could be consistently observed in heavily infected rats.     

Structural bases for the specific interactions between the E2 and E3 components of the Thermus thermophilus 2-oxo acid dehydrogenase complexes

Pyruvate dehydrogenase (PDH), branched-chain 2-oxo acid dehydrogenase (BCDH) and 2-oxoglutarate dehydrogenase (OGDH) are multienzyme complexes that play crucial roles in several common metabolic pathways. These enzymes belong to a family of 2-oxo acid dehydrogenase complexes that contain multiple copies of three different components (E1, E2 and E3). For the Thermus thermophilus enzymes, depending on its substrate specificity (pyruvate, branched-chain 2-oxo acid or 2-oxoglutarate), each complex has distinctive E1 (E1p, E1b or E1o) and E2 (E2p, E2b or E2o) components and one of the two possible E3 components (E3b and E3o). (The suffixes, p, b and o identify their respective enzymes, PDH, BCDH and OGDH.) Our biochemical characterization demonstrates that only three specific E3*E2 complexes can form (E3b*E2p, E3b*E2b and E3o*E2o). X-ray analyses of complexes formed between the E3 components and the peripheral subunit-binding domains (PSBDs), derived from the corresponding E2-binding partners, reveal that E3b interacts with E2p and E2b in essentially the same manner as observed for Geobacillus stearothermophilus E3*E2p, whereas E3o interacts with E2o in a novel fashion. The buried intermolecular surfaces of the E3b*PSBDp/b and E3o*PSBDo complexes differ in size, shape and charge distribution and thus, these differences presumably confer the binding specificities for the complexes.